A putative gene, denoted ysbC, was previously identified by genome sequencing of a Lactococcus lactis strain, but the gene was not annotated, and no function of the predicted encoded polypeptide was identified (WO 200177334, Bolotin et al., 2001, The Complete Genome Sequence of the Lactic Acid Bacterium Lactococcus lactis ssp. Lactis IL1403, Genome Res. 11:731). The amino acid sequence of the predicted YsbC protein is available from the database GeneSeqP, accession number: ABB55104.
There is growing concern in the public, as well as in the scientific communities, about the use of antibiotics in molecular biology. In particular, there is concern about the cultivation of recombinant cells carrying antibiotic genetic markers, mostly due to the perceived potential risk for lateral transfer of such markers into nature. It is therefore of interest to identify new non-antibiotic marker genes to substitute the classical antibiotic resistance encoding selection, counter-selection, screening, and bi-directional selection markers in molecular biology.
The present invention relates to an orotate transporter polypeptide derived from a Lactococcus lactis strain. The orotate transporter is encoded by the ysbC gene, an open reading frame, which was predicted but not annotated in an earlier genome sequencing project.
A search in the public databases for any polypeptides having amino acid sequence homology to the orotate transporter of the present invention, revealed that the closest polypeptide had less than 35% sequence identity, and it was completely unrelated to the orotate transporter of the present invention. Consequently, the ysbC-encoded orotate transporter represents a completely pioneering new class of molecules.
The newly identified orotate transporter was shown to be functional in both Gram-positive and Gram-negative bacterial cells, and it was shown that ysbC is ideally suited as a versatile non-antibiotic marker gene, suitable for use in selection, counter-selection, screening, and bi-directional selection protocols, as exemplified below.